Gap junctions allow diffusion-driven intercellular transfer of small macromolecules and ions, which, in turn, regulate metabolic and ionic homeostasis, growth and differentiation. Gap junctions synchronize electrical activity necessary for a normal heart rhythm and whose abnormality is the leading cause of sudden death. Abnormal gap junctional communication is also associated with other diseases, including cancer and neurodegenerative diseases. Recently, we have shown that the cell plasma membrane contains hemichannels in the non-junctional regions and these hemichannels "sense" extracellular physiological calcium. We also showed that these hemichannels i) allow direct communication between the cytoplasm and the extracellular space, ii) alter cellular ionic homeostasis, iii) regulate cell volume, and iv) induce death of metabolically impaired cells (Quist et al, 2000). Hence, hemichannels may play a significant role in pathological situations where there is an increased hemichannel activity. Hemichannels open in response to reduced extracellular Ca2+ in the physiological range or in response to membrane depolarization. Open hemichannels alter cellular ionic homeostasis, potentially increasing Ca2+ and reducing pH, both of which together will inhibit gap junctional communication (GJIC). Hemichannels thus could modulate both cell-cell and cell-extracellular communications. Having determined a definite physiological role of hemichannels, it is now important to understand the mechanism of calcium sensing, their binding epitope(s) on the extracellular regions and their effects on hemichannel' s 3D conformation. We propose to study the 3D molecular structure, activity and ligand-induced conformations of hemichannels using atomic force microscopy (AFM), electrical recording, biochemical and molecular biological techniques. The specific aims are: 1) imaging 3D molecular structure of hemichannels in reconstituted bilayer membrane and in the plasma membrane preparations from connexin-expressing cells, 2) examining the effects on hemichannel 3D conformations of perturbations (such as calcium and membrane potentials) which regulate the functional states (open vs. closed states), and 3) in order to determine putative sites of Ca2+-sensors, comparing the molecular structure of hemichannels isolated after site-directed mutagenesis of extracellular epitopes on Cx43, or wild type hemichannels using antibody-connexin affinity by single molecule force spectroscopy. An understanding of the activity and role of hemi-junctional activity could provide important information about normal and abnormal physiological conditions.